Polyribozyme capable of conferring on plants resistance to viruses and resistant plants producing this polyribozyme

ABSTRACT

The invention relates to a nucleic acid sequence, called “polyribozyme”, which has an endoribonuclease activity and is capable of inactivating the gene for the capsid protein of a virus, characterized in that it comprises: 
     i) a sequence complementary to at least a part of the gene or its transcript or to its replication intermediates and, includes at distinct sites in this complementary sequence: 
     ii) a plurality of ribozyme catalytic regions; 
     iii) and, optionally, one or more sequences non-complementary to the transcript of the said gene, the said non-complementary sequence(s) being inserted between two consecutive bases of the complementary sequence.

This application is a continuation of U.S. Ser. No. 08/507,426, now U.S.Pat. No. 6,265,634, filed Oct. 25, 1995, which is a §371 National Stageof PCT International Application No. PCT/FR94/00216, filed Feb. 25,1994, which claims, priority of French Application No. 93 02269 filedFeb. 26, 1993.

The present invention relates to a nucleotide sequence, called“polyribozyme”, capable of conferring on plants resistance to viruses,as well as a process for making the plants resistant. The invention alsorelates to the plants expressing the polyribozyme.

Several approaches have been developed to confer on cultivated plantsresistance to viruses by integrating into the genome of the plants viralnucleic acid sequences: the gene for the capsid protein, the genes fornon-structural proteins, anti-sense viral RNA sequences and RNAs ofsatellite viruses (see, for example, Cuozzo et al., 1988, Bio/Technology6, 549-557; Rezaian et al., 1988, Plant. Mol. Biol., 11, 463-471;Harrison et al., 1987, Nature 328, 797-802).

These publications report the production of partial resistances ortolerances. Nonetheless, in most cases there are delayed symptoms orattenuated symptoms but not complete resistance.

Furthermore, some of these procedures, for example those employing theRNAs of satellite viruses, can give rise to new problems. For example, asatellite virus which reduces symptoms in one species may become lethalfor another species. Moreover, mutations in the nucleotide sequences ofthe satellite virus introduced into the plants may increase the severityof the infection instead of diminishing it.

Similarly, the use of the capsid protein to confer resistance hasdisadvantages. For example, the capsid protein of a particular strain ofthe virus does not necessarily protect the plant against an infection byanother strain of the virus. It is difficult to use the degree ofhomology of the amino acid sequence of the capsid protein betweendifferent viruses or between different strains to predict the degree oftolerance allowed by the expression of the protein. Furthermore, theexpression of capsid proteins to protect against viral infectionpresents the risk of inducing heteroencapsidation between the capsidprotein expressed in the plant and other viruses infecting thetransgenic plant. Although it has never been demonstrated for transgenicplants, this heteroencapsidation has already been observed between twostrains of BYDV and between ZYMV and PRSV.

The use of ribozymes has also been considered for conferring on plantsresistance to viruses. Ribozymes are RNA molecules which act as enzymesby specifically catalysing the cleavage of the target RNA. The firstexperiments with ribozymes in plant cells were described in the patentapplication EP-A-321021. Since then, several authors have tried tooptimise the structure of the ribozyme and the operating conditions inorder to obtain efficient cleavage of the viral RNA.

For example, Lamb and Hay (J. Gen. Virol., 1990, 71:2257-2264) havedemonstrated the in vitro cleavage by mono-ribozymes of the RNA of thePotato Leaf Roll virus (PLRV) in regions coding for the RNA polymeraseand the capsid protein. However, the in vitro cleavage reaction onlyoccurs at 40° C.; it has not been possible to observe any reaction atall at 0° C. Plants are usually cultivated between 10 and 30° C.,depending on the species. Thus, for in vivo use, Lamb and Hay suggestthat the length of the complementary arms be increased. But, if the armsare too long, the formation of a stable duplex between the target RNAand the ribozyme can be caused, preventing the dissociation of theribozyme and making it incapable of catalysing another cleavagereaction. Furthermore, depending on the length and sequence of thecomplementary arms, the ribozyme itself may form secondary structureswhich diminish its cleavage activity.

Edington and Nelson (“Gene Regulation: Biology of Antisense RNA and DNA:Ed. ERICKSON and IZANT, Raven Press Ltd, New-York, 1992) have describedthe in vitro and in vivo use of mono- ribozymes to inactivate thepolymerase gene of the Tobacco Mosaic virus (TMV). They observed thatthe ribozymes exhibited a very different behaviour depending on whetherthey were used in vitro or in vivo. The activity of a ribozyme in vitrocan not thus be used to predict the activity of the same ribozyme invivo. For example, in vitro cleavage appear to be of low efficiency andrequires a ribozyme concentration 20 times higher than the concentrationof the TMV genomic RNA. On the other hand, in an in vivo experimentusing tobacco protoplasts infected by TMV, the ribozyme suppresses 90%of the multiplication of the viral RNA. It is interesting to note thatthe anti-sense RNA used as control only inhibits 20% of the viralmultiplication. These workers also refer to the studies of Gerlach etal. who made use of a polyribozyme targeted against the gene for thepolymerase of TMV. This polyribozyme did not function in vitro owing tothe length of the duplex formed between the ribozyme and the target RNA.On the other hand, in vivo, this polyribozyme cleaved the substrate.Transgenic tobacco plants expressing either the monoribozyme or thepolyribozyme have shown a delay of symptoms after infection by the TMV.Complete resistance, i.e. the definitive absence of symptoms, is notdescribed. The authors conclude that the parameters such as the optimallength of the complementary arms, the choice of the target sequence andthe choice of the promoter, enabling possible problems of“compartment-alisation” of the ribozymes to be overcome, must bedetermined by experiment.

EP-A-0421376 describes ribozymes directed against a non-coding RNAsequence of CMV. WO-A-9213090 describes the inactivation of the RNA ofthe capsid protein of the CMV by the introduction of a heterologoussequence within the sequence using a monoribozyme of the “Group Iintron” type. None of these documents describes the production ofcomplete resistance to the CMV.

The technical problem which the present invention proposes to resolve isto provide a reliable agent, devoid of disadvantages, for conferring onplants resistance to viruses.

The present inventors have resolved this problem by the conception anduse of a polyribozyme directed against the capsid protein of a virus.This polyribozyme is capable of inactivating the gene coding for thisprotein, and of thus conferring complete resistance to viruses.

The efficiency of the polyribozyme of the invention is surprising in thelight of the mediocre results obtained in the prior art with theanti-sense sequences of the gene for the capsid protein, since each ofthese procedures involves an inactivation of the corresponding RNA. Inaddition, several authors had advised against the use of trans actingpolyribozymes because the ribozymes are unable to function independentlyof each other and because catalytic regions having identical sequencessometimes have a tendency to hybridize to each other, which leads toinactive structures (see, for example, Taira, HFSP. Workshop “RNA-Editing—Plant Mitochondria”, Abstract Book, Berlin, Sep. 15-20, 1992).The results obtained according to the invention are unexpected in viewof the target selected, on the one hand, i.e. the capsid protein and, onthe other hand, the method used to inactivate the target, i.e. apolyribozyme.

In addition to the efficiency of the inactivation, the polyribozymes ofthe invention possess a number of advantages in comparison with knownprocedures:

The ribozymes function as enzymes, catalysing the cleavage of severalviral RNAs specifically without modification of structure. Thisenzymatic cleavage leads to the destruction of all of the viral RNAswhereas the expression of the capsid protein which inhibits viralinfection functions as an inhibitor of viral multiplication.

The ribozymes are non-coding RNA molecules which can not induceheteroencapsidations or generate new viral strains.

Whereas the specificity of the tolerance induced by the capsid proteinis difficult to predict, ribozymes can be constructed in order to cleavespecifically one or more viral strains, or several related viruses ifthe complementary arms correspond to the regions of homology conservedbetween the different strains or between the different related viruses.

In order to have a complete understanding of the invention, it will beuseful to specify certain facts concerning ribozymes in general. Aribozyme is an RNA molecule which, by virtue of its sequence andsecondary structure, possesses an endoribonuclease activity whichenables it, when it hybridizes with a second molecule of complementaryRNA, to cleave this second RNA. This latter is hence a “substrate” forthe ribozyme.

The ribozyme has two essential parts:

(i) a sequence, which will be called “complementary sequence” in whatfollows, and which is selected so that, it is complementary to thesubstrate which it is desired to cleave, this enabling the two moleculesto hybridize;

(ii) and a catalytic region which has a conserved sequence irrespectiveof the substrate selected and which does not take part in thehybridization with the substrate on account of its secondary structurewhich is in the form of a “loop”.

Usually, the catalytic region is located within the complementarysequence, one part of the complementary sequence thus being situated atthe 5′ of the catalytic region and the other part at the 3′. Thesefragments of the complementary sequence on each side of the catalyticregion are often called “hybridizing arms”.

The object of the present invention is a polyribozyme. Moreparticularly, it is a nucleic acid sequence, called “polyribozyme”,which has an endoribonuclease activity and is capable of inactivatingthe gene for the capsid protein of a virus, characterized in that itcomprises:

i) a sequence complementary to at least a part of the gene or itstranscript or its replication intermediates and, included at distinctsites in this complementary sequence:

ii) a plurality of ribozyme catalytic regions;

iii) and, optionally, one or more sequences not complementary to thetranscript of the said gene, the said non-complementary sequence(s)being inserted between 2 consecutive bases of the complementarysequence.

The term “polyribozyme” in the context of the present invention means anRNA molecule constituted by a head-to-tail series of ribozymes, theribozyme thus being the unit motif of the polyribozyme. In other words,it is a series of catalytic regions connected together by hybridizingarms, the total length of these arms constituting the complementarysequence. The polyribozyme normally acts as a “uni-molecule” against asingle transcript, i.e. the cleavage sites of each of the catalyticregions are located on the same transcript: the capsid protein. Thepolyribozyme of the invention may also comprise, in addition to the 2essential parts [(i), complementary sequence] and [(ii), catalyticregions] described above, one or more sequences (iii) non-complementaryto the substrate. The nature and function of these non-complementarysequences will be described in detail hereafter.

Of the 2 essential parts of the polyribozyme, the complementary sequenceis that which determines the substrate. In the case of the presentinvention, it is a sequence complementary to the gene for the capsidprotein of a virus, or to a fragment of this gene. When the virus is anRNA (+) virus, the genes of which serve directly as mRNA, thecomplementary sequence is really complementary to the gene. In othercases, it is complementary to the transcript of the gene. It may also becomplementary to a replication intermediate.

The complementary sequence may hybridize with the entire length of thecapsid gene. In this case, the total length of the complementarysequence varies as a function of the length of the capsid gene inquestion. On the other hand, the complementary sequence may hybridizewith only a fragment of the gene. The fragment in question must be longenough to allow the inclusion of at least two catalytic regions in thecorresponding sequence of the polyribozyme. In general, the length ofthe complementary sequence, not counting the catalytic regions (i.e. thesum of the hybridizing arms), may vary from about 40 to 2000 bases. Alength of 400 to 1000 is preferred, very many viruses having a gene forthe capsid protein of about 1000 bases (for example, CMV, PLRV).

The term “complementary” in the context of the invention means asufficiently high degree of complementarity to allow stablehybridization between the polyribozyme and this substrate, and theefficient cleavage of the substrate. When the polyribozyme does notcontain a sequence of type (iii), i.e. “non-complementary”, the degreeof complementarity is usually 100%. The presence of a certain number ofmismatches in the sequence, for example up to 10%, may be toleratedprovided that that does not prevent the hybridization and cleavage ofthe substrate.

The (ii) part of the polyribozyme, i.e. the catalytic region, is derivedfrom any type of suitable ribozyme, for example “hammer head”, “hairpin”or “group I intron”. One and the same polyribozyme may contain catalyticregions derived from different types of ribozymes, for example, “hammerhead” and hairpin”. Catalytic regions are preferably derived fromribozymes of the “hammer head” type, the consensus structure of which isillustrated in the FIGS. 1A, B, C and D. These ribozymes are describedin detail in the patent application EP-A-321021 and WO-A-9119789.

Although the catalytic regions illustrated in FIG. 1 have a conservedstructure and sequence, it has been observed that some nucleotides maybe deleted, inserted, substituted or modified without prejudice to theactivity of the ribozyme. The invention comprises the use of thesemodified catalytic regions in the polyribozyme provided that theircatalytic activity is conserved. This activity can be verified by usingthe tests described below.

For example, one or more nucleotides of the catalytic region IIillustrated in FIG. 1A may be replaced by nucleotides containing basessuch as adenine, guanine, cytosine, methylcytosine, uracil, thymine,xanthine, hypoxanthine, inosine or other methylated bases. The“conserved” bases C-G which together form the first base pair of thecatalytic loop, can be replaced by U-A (Koizumi et al., FEBS Letts. 228,2, 228-230, 1988).

The nucleotides of the catalytic region illustrated in FIG. 1 can alsobe modified chemically. The nucleotides are composed of a base, a sugarand a monophosphate group. Each of these groups can thus be modified.Such modifications are described in “Principles of Nucleic AcidStructure” (Ed. Wolfram Sanger, Springer Verlag, N.Y., 1984). Forexample, the bases may bear substituents such as halogeno, hydroxy,amino, alkyl, azido, nitro, phenyl groups, etc. The sugar moiety of thenucleotide may also be subjected to modifications such as thereplacement of the secondary hydroxyl groups by halogeno, amino or azidogroups or even to 2′ methylation.

The phosphate group of the nucleotides may be modified by thereplacement of an oxygen by N, S or C, giving rise to a phosphoramidate,phosphorothioate and phosphonate, respectively. These latter may exhibituseful pharmacokinetic properties.

The bases and/or the nucleotides of the catalytic region may also bearsubstituents such as amino acids, for example, tyrosine or histidine.

It has also been observed that additional nucleotides may be inserted atcertain sites of the catalytic region without prejudice to the activityof the ribozyme. For example, an additional base selected from among A,G, C or U may be inserted after A¹ in FIG. 1A or 1B.

According to a variant of the invention, the ribozyme may comprise ascatalytic region one or more structures such as those illustrated inFIG. 1D. This structure, called “minizyme”, is described in theinternational patent application WO-A-9119789. It represents a catalyticregion of the “hammerhead” type, the “loop” of which has been replacedby a “P” group. P may be a covalent link between G and ¹A, one or morenucleotides (RNA or DNA, or a mixture, or even derivatives describedabove) or any atom or group of atoms other than a nucleotide which doesnot affect the catalytic activity. When P represents a plurality ofnucleotides, it may contain internal base pairings. The sequence and thenumber of nucleotides constituting the group “P” is not critical and mayvary from 1 to 20 nucleotides for example, and preferably from 1 to 6.It is preferable to select a sequence lacking internal base pairings ofthe Watson-Crick type.

The catalytic activity of the polyribozymes of the invention may beverified in vitro by placing the polyribozyme, or a sequence which aftertranscription will give rise to the polyribozyme, in contact with thesubstrate, followed by demonstration of the cleavage. The experimentalconditions for the in vitro cleavage reaction are the following: atemperature comprised between 4 and 60° C., and preferably between 20and 55° C., a pH comprised between about 7.0 and 9.0, in the presence ofdivalent metals, such as Mg²⁺, at a concentration of 1 to 100 mM(preferably 1 to 20 mM). The polyribozyme is usually present in anequimolar ratio with the substrate, or in excess. The in vitro cleavagereactions are advantageously carried out according to the proceduredescribed by Lamb and Hay (J. Gen. Virol., 1990, 71, 2257-2264). Thisarticle also describes suitable conditions for in vitro transcriptionfor the production of ribozymes from oligodeoxyribonucleotides insertedinto plasmids.

The in vivo cleavage conditions are those existing naturally in thecell.

The “hammerhead” ribozymes cleave the substrate immediately downstreamfrom a “target” site XXX, preferably XUX, in which X represents one ofthe 4 bases A, C, G, U and U represents uracil. One particularlypreferred target sequence is XUY in which Y represents A, C or U and Xisoften G, for example, GUC. Other target sites are possible, but lessefficient, for example CAC, UAC and AAC. In the case of the ribozymes ofthe “hairpin” type, a preferred target sequence is AGUC.

These target sequences are important in the construction and functioningof the polyribozymes, not only because they indicate the positions ofcleavage of the substrate but also because they define the position atwhich the catalytic region must be inserted in the complementarysequence. In fact, each catalytic region of the polyribozyme must besituated at a site in the complementary sequence which corresponds to aXUX site of the transcript. For example, if one XUX site is situated atposition 108 of the gene for the capsid protein and another at position205, a catalytic region is inserted at the corresponding position at 108in the complementary sequence and another at 205.

The motif XUX is a motif which occurs very frequently in the RNAsequences. For example, on average there is a GUC motif every 64 basesin a sequence having a random and equal distribution of bases. Thissignifies that the substrate usually contains a plurality of XUXcleavage sites. As indicated above, the catalytic regions of thepolyribozymes are situated at positions of the complementary sequencewhich correspond to the XUX sites. However, it is not necessary toinclude a catalytic region for each XUX target sequence of the substratein order to obtain an efficient cleavage according to the invention.According to the invention, an efficient cleavage is obtained when thepolyribozyme contains at least 2 catalytic regions. The total number ofcatalytic regions included in the complementary sequence is equal to orsmaller than the total number of XUX sites present in the gene. Thepolyribozyme of the invention may thus contain a very variable number ofcatalytic regions. For example, in the case of CMV, the polyribozyme maycontain from about 2 to about 11 or 12 catalytic regions, when thetarget sequence is GUC. In the case in which it is decided to include asmaller number of catalytic regions in the complementary sequence thanthe number of XUX sites in the substrate, the choice of the sitesselected may be made by respecting the following criteria:

a) the distance between the 2 XUX sites targeted and, consequently,between 2 catalytic regions in the polyribozyme must be long enough toenable the hybridizing arms of the polyribozyme situated between thecorresponding catalytic regions to hybridize with the substrate in astable manner and to prevent the catalytic regions hybridizing withthemselves. A distance of at least 8 bases, and preferably at least 14bases, for example about 20 bases is particularly advantageous. Ofcourse, this criterion must only be taken into consideration when thesubstrate contains XUX sites very close together. Otherwise, if the XUXsites of the substrate are separated from each other by more than 8 to20 bases, this selection criterion is not important.

b) the XUX sites targeted are preferably situated in a part of the genefor the capsid protein which does not have significant secondarystructure. This facilitates the access of the polyribozyme to thesubstrate and increases its efficacy.

c) the XUX sites targeted may form part of the regions of homologyconserved between different strains of one and the same virus, orbetween different related viruses. The polyribozymes constructed byrespecting this criterion may cleave specifically several viral strainsor several related viruses. For example, the central region of the genefor the capsid protein of the PLRV is highly conserved compared withsequences of the capsid proteins of the related viruses BWYV and BYDV.The XUX sites, and particularly GUX within this central region, thusconstitute preferred sites for a polyribozyme according to this variantof the invention.

Also, by way of example, the 5′ end (over a length of about 100 bases)of the sequence of the capsid protein of the CMV is highly conservedbetween the strains I17F, FNY, M, I, O, Y, D and C. At position 84within this conserved sequence there is a conserved GUC site in all ofthese strains.

According to this variant of the invention, a polyribozyme capable ofinactivating several strains of the CMV comprises among its catalyticregions one catalytic region which is situated at the site of thecomplementary sequence corresponding to the position 84. (see exampleshereafter).

d) another selection criterion of the XUX sites targeted is the absenceof homology with endogenous genes of the plant to be transformed. Infact, although they are rare, some viruses possess sequences which finda homology in the genome of plants. It is thus important to avoid XUXsites situated within such a sequence.

According to a particularly preferred embodiment of the invention, thepolyribozyme may comprise, in addition to the 2 essential parts (i) and(ii) described above, a 3rd constituent (iii) which is one or moresequences non-complementary to the gene for the capsid protein of thevirus. Like the catalytic regions, these non-complementary sequences areinserted at distinct sites of the complementary sequence, thecomplementarity being interrupted by the insertion. Surprisingly, it wasobserved by the inventors that the presence of such non-complementarysequences within the hybridizing arms of the polyribozyme does notprevent the hybridization of the polyribozyme with the substrate, and insome cases can even improve the efficiency of the cleavage reaction.

These non-complementary sequences are inserted between 2 consecutivebases of the complementary sequence, the non-complementary sequence thusforming a colinear insertion with the complementary sequence. In thiscase, the polyribozyme has the structure:

((hybridizing arm-catalytic region-hybridizing arm)−(non-complementarysequence)_(n))_(p)

in which n=0 or 1, and p>1.

As an example of this embodiment of the invention, mention may be madeof a polyribozyme composed of a sequence of ribozymes, the hybridizingarms of which are complementary to distinct fragments, consecutive andadjoining, of the substrate and which are connected together bynon-complementary sequences. In other words, the aggregate of thehybridizing arms in such a structure reconstitute the sequencecomplementary to the gene for the capsid protein.

The presence of non-complementary sequences in the polyribozymesignifies that the distance between two catalytic regions of thepolyribozyme is greater than the distance between two corresponding GUCsites in the substrate. According to this variant of the invention, thelength of the hybridizing arms located on each side of a catalyticregion must be at least 4 bases, and preferably at least 8 bases on eachside and may be as many as 800 to 1000 bases.

The nature of the non-complementary sequence(s) may be very variabledepending on its (their) function. There may be sequences which have a“padding” function, i.e. which serve to increase the distance betweentwo catalytic regions of the polyribozyme, when the corresponding twoXUX sites of the substrate are relatively close to each other. In thismanner, the formation of inactive duplexes between two neighbouringcatalytic regions can be avoided. It is also possible to use asnon-complementary sequences, sequences which have a defined secondarystructure, which may have the effect of preventing a polyribozyme ofconsiderable length, for example one with more than 800 bases, fromrefolding on itself in an inactive secondary structure. As an example ofthis type of structure, mention may be made of a ribozyme renderedinactive by the deletion of one or more essential bases. This mode ofembodiment of the invention is exemplified by the polyribozyme 136described in the examples below.

The non-complementary sequence of the polyribozyme may also have aprecise function, for example, it may be constituted by a codingsequence which can be used to select transformants or also a sequencecontaining a ribozyme which acts on a substrate other than the capsidprotein or which is cis acting on a part of the polyribozyme. Generallyspeaking, the non-complementary sequence does not code for a protein. Itmay also contain multisites for cloning. The non-complementary sequenceusually has a length comprised between 2 and 500 bases, for example 20to 100 bases. When there is a plurality of complementary sequences, theymay together constitute up to about 90% of the length of thepolyribozyme, for example 50%.

The polyribozyme of the invention is usually constituted of RNA.Nonetheless,it is possible to replace some parts of the polyribozyme byDNA, for example the hybridizing arms or parts of these arms, or also apart of the catalytic region, in particular the “loop”, provided thatthe catalytic activity is maintained (see, for example, the substitutionof the RNA by DNA described in the international patent applicationWO-9119789).

The polyribozyme of the invention can be constructed to inactivate anyviral capsid protein. The capsid protein is the protein sub-unit, codedby the viral genome, which makes up the polymeric capsid. The capsid iscomposed of a succession of these identical protein sub-units whichline-up along the nucleic acid. The spatial arrangement of the capsidsub-units gives rise to either a helicoidal or an icosahedric structure,according to the virus. The invention concerns polyribozymes directed tothe capsid proteins of viruses having either helicoidal particles, oricosahedric particles, as well as those having an envelope. The envelopeis a lipoprotein membrane surrounding the nucleocapsid.

As an example of a suitable virus, mention may be made of a virusselected from the following groups: the caulimoviruses, for example theCauliflower Mosaic Virus (CaMV); the Geminiviruses, for example theMaize Streak Virus (MSV); the Reoviridae, for example the Wound TumorVirus (WTV); the Rhabdoviridae, for example the Potato Yellow DwarfVirus (PYDV), the Tomato Spotted Wilt Virus (TSWV); the Tobamoviruses,for example the Tobacco Mosaic Virus (TMV); the Potexviruses, forexample the Potato Virus X (PVX); the Potyviruses, for example thePotato Virus Y (PVY); the Carlaviruses, for example the Carnation LatentVirus (CLV); the Closteroviruses, for example the Beet Yellow Virus(BYV); the Tobraviruses, for example the Tobacco Rattle Virus (TRV); theHordei-viruses, for example the Barley Stripe Mosaic Virus; theTymoviruses, for example the Turnip Yellow Mosaic virus (TYMV); theLuteoviruses, for example the Barley Yellow Dwarf Virus (BYDV) or thePotato Leaf Roll Virus (PLRV); the Tombusviruses, for example the TomatoBushy Stunt Virus (TBSV); the Sobemoviruses, for example the SouthernBean Mosaic Virus (SBMV); the Tobacco Necrosis virus (TNV); theNepoviruses, for example the Tobacco Ring Spot Virus (TRSV); theComoviruses, for example the Cow Pea Mosaic Virus (CPMV); the PeaEnation Mosaic Virus (PEMV); the Cucumoviruses, for example the CucumberMosaic Virus (CMV); the Bromoviruses, for example the Brome Mosaic Virus(BMV); the Ilarviruses, for example the Tobacco Streak Virus (TSV). thesequences of these proteins are known (see for example the numerousliterature references cited in the monograph: “Eléments de VirologieVégétale”, Pierre Cornuet, I. N. R. A. Paris, 1987, ISBN:2-85340-808-6).

According to a particularly preferred variant, the capsid protein isthat of the Cucumber Mosaic Virus (CMV). The Cucumber Mosaic Virus is avirus belonging to the group of the Cucumoviruses which are of greatagronomic importance since more than 750 species of plants may beinfected by the CMV. The CMV is a multi-component virus composed oficosahedral particles containing three genomic RNAs (RNAs 1 to 3) and asubgenomic RNA (RNA 4). The RNA 3 contains a copy of the gene for thecapsid protein; however, the subgenomic RNA4, which is derived from RNA3, serves as matrix for the synthesis of the capsid protein. Thedifferent strains of CMV are divided into two groups:

the sub-group I which comprises the strains C, D, FNY, Y, I17F and Chi;

the sub-group II to which the strains Q and WL belong.

The comparison of the amino acid sequences of the capsid proteins of theCMV strains belonging to the same sub-group shows a homology of 95%. Thesequence homologies between the sub-groups I and II are lower, of theorder of 80%.

The polyribozymes of the invention directed against the capsid proteinof the CMV (strain I17F) have been found to be extremely efficient ininactivating the different strains of the CMV, and have resulted incomplete resistance of the transformed plants.

In addition to the polyribozymes, the invention also relates to aprocess for making a plant resistant to a virus, characterized by theintroduction into the plant of a polyribozyme or a sequence coding for apolyribozyme such as described above.

Usually, the introduction of the polyribozyme into the plant isperformed by genetic transformation, a DNA sequence coding for thepolyribozyme thus being integrated stably into the genome of the plant.

All of the known means for introducing foreign DNA into plants may beused, for example Agrobacterium, electroporation, protoplast fusion,bombardment with a particle gun, or penetration of DNA into cells suchas pollen, the microspore, the seed and the immature embryo, viralvectors such as the Geminiviruses or the satelite viruses. Agrobacteriumtumefaciens and rhizogenes constitute the preferred means. In this case,the sequence coding for the polyribozyme is introduced into a suitablevector together with all of the regulatory sequences necessary such aspromoters, terminators, etc. . . . as well as any sequence necessary forselecting the transformants.

The invention also relates to the transgenic plants obtained by theprocess. More particularly, it relates to transgenic plants resistant toa virus, characterized in that they contain in their genome a sequencewhich, after transcription, gives rise to a polyribozyme according tothe invention .

In the context of the invention, the term “complete resistance”signifies a complete absence of symptoms; “tolerance” signifies that theplant is infected, i.e. it shows symptoms, but subsequently recovers.The term “sensitive” signifies that the plant exhibits symptoms andreplicates the virus. The expression “resistant type” refers to the sumof the completely resistant plants and the tolerant plants.

The “resistant” nature of the transgenic plants of the invention can betested in the following manner: a self-fertilization, or a cross with anon-transformed genotype, is carried out on a primary descendant toobtain T₁. Subsequently, T₁ plants is inoculated with the virus inquestion. According to the invention, after self-fertilization 75% ofthe T₁ are completely resistant. In the case of a cross with anon-transformed genotype, 50% of the plants are completely resistant(these figures are obtained, according to the invention, by testing awhole population of plants which had been subjected to a transformationand regeneration procedure. It is to be noted that only 75% of theseplants are transformed).

The transformed nature of a plant can be verified by performing a“Southern blot” analysis and the expression of the sequence introducedby the transformation is verified by carrying out a “Northern blot”analysis. These analyses are described in the examples which follow.

The methods of transformation and regeneration of plants known in theprior art lend themselves perfectly to the production of transgenicplants protected by the polyribozyme of the invention. As an example,mention may be made of the method of transformation and regeneration ofthe melon described in the patent application No. EP-A-0412912.

The transgenic plants resistant to the CMV are particularly preferred,for example the melon, the cucumber, the courgette, the tomato, thepepper, the bean.

Various aspects of the invention are illustrated in the Figures:

FIGS. 1(A-D) shows the preferred structures of the catalytic regions ofthe polyribozyme of the invention, these regions being surrounded oneach side by a sequence complementary to a part of the capsid protein ofa virus:

(i) in FIG. 1A: X represents A, G, C or U; each X being identical ordifferent; n+n′≧6, n and n′ being identical or different; (*) representsa hydrogen bond between complementary ribonucleotides; X′ and X″represent oligoribonucleotides which are complementary to each otherover at least a part of their length and which may possibly be connectedto each other by at least one nucleotide, thus forming a loop. Anadditional nucleotide selected from A, G, C or U may be inserted afterA′. The catalytic region of the ribozyme is represented by part (II) ofFIG. 1A, and the hybridizing arms by part (I).

(ii) in FIG. 1B: X, (*), n, n′ and A′ have the same meaning as in FIG.1A. M and M′≧1 and are identical or different. B represents a bond, abase pair, a ribonucleotide or an oligoribonucleotide containing atleast 2 ribonucleotides.

(iii) FIG. 1C SEQ. I.D. NO. 14 represents a preferred model of ribozymes(Haseloff and Gerlach, 1988) The RNA substrate may have any sequence (X)around the GUC cleavage site complementary to the ribozyme. The arrowindicates the cleavage site. The conserved bases are shown in black.

(iv) FIG. 1D represents the structure of a ribozyme (called “minizyme”),the loop of the catalytic region of which is replaced by an element “P”.P may be at least 1 nucleotide (ribonucleotides, deoxyribo-nucleotides,derivatives or a mixture), provided that the ribonucleotides of the “P”group are not base paired by “Watson-Crick” base pairings when thesequences (X′)_(n) and (X)_(n′) and P are constituted exclusively ofribonucleotides. “P” may also be a bond or any atom or group of atomswhich do not affect the catalytic activity of the ribozyme. X has thesame meaning as FIG. 1A.

FIG. 2 shows the sequence of the capsid protein of the CMV (I7F)SEQ.I.D. NO. 5, the GUC sites being underlined.

FIG. 3 presents the structures of the oligodeoxyribonucleotides A SEQ.I.D. NO. 6; SEQ. I.D. NO. 7, B SEQ. I.D. NO. 8; SEQ. I.D. NO. 9, C SEQ.I.D. NO. 10; SEQ. I.D. NO. 11, E SEQ. I.D. NO. 12; SEQ. I.D. NO. 13,used for the directed mutagenesis experiments for the purpose ofintroducing the catalytic site of the TobRSV at different sites in thesequence of the capsid protein of the CMV (shown hybridized with the DNAmatrix sequence).

FIG. 4 illustrates the structure of the genes constructed in order toinduce resistance to the CMV:

(i) capsid protein

(ii) polyribozyme 136: sequence complementary to the capsid proteinbearing 2 ribozymes;

(iii) polyribozyme 161: sequence complementary to the capsid proteinbearing 3 ribozymes;

(iv) polyribozyme 163: fragment complementary to the capsid proteinbearing 3 ribozymes;

(v) polyribozyme 165: sequence complementary to the capsid proteinbearing 4 ribozymes;

FIG. 5 shows the Northern blot analysis of the transgenic melon plants(primary transformants, T1 and T2 descendants in some cases) expressingthe polyribozyme 136:

T0: primary transformant;

T1: T1 descendant;

T2: T2 descendant;

A: line 141.1;

NT: untransformed control.

FIG. 6 shows the Southern analysis of the transgenic melon plants(primary transformants, T1 and T2 descendants in some cases) expressingthe polyribozyme 136:

T0: primary transformant;

T1: T1 descendant;

T2: T2 descendant;

NT: untransformed control

FIG. 7 shows the Southern blot analysis of the transgenic melon plants(primary transformants, T1 and T2 descendants in some cases) expressingthe gene for the capsid protein:

T0: primary transformant;

T1: T1 descendant;

T2: T2 descendant;

A: line 88.105;

B: line 159.8

NT: untransformed control.

FIG. 8 shows the Western blot analysis of the transgenic melon plants(primary transformants) transformed by pBIOS135:

A, B, C, D and E: primary transformants of 5 lines;

NT: untransformed control;

R: reconstruction with 20 ng of CMV.

FIG. 9 shows the development of the symptoms of the CMV with time in thelines transformed by pBIOS 135:

D: day;

: plants without symptoms

: tolerant plants

: sensitive plants.

FIG. 10 shows the development of the symptoms of the CMV with time inthe lines transformed by pBIOS 135:

D: day;

: plants without symptoms

: tolerant plants

: sensitive plants.

EXAMPLES

The following examples describe the construction of 4 polyribozymescontaining 3 or 4 ribozymes composed of the Hammerhead consensusstructure of 24 bases (FIG. 1) and arms complementary to the sequence ofthe capsid protein of the CMV (I17F strain) of different sizes. Thenumbering of the nucleotide sequence of the capsid protein shown inthese examples corresponds to those used in the patent applicationEP-A-0412912.

Each of these ribozymes cleaves a different GUC sequence along thesequence of the gene for the capsid protein of the CMV. The structure ofthese constructs is illustrated in FIG. 4:

The polyribozyme 136, 1074 bases long, contains the ribozymes A(position 84) and B (position 108) and the ribozyme C* (position 204),from which a G and an A (positions 20 and 21) have been deleted, theribozymes being surrounded by complementary arms of the followinglengths:

82 nucleotides from the 5′ end to the ribozyme A;

22 nucleotides between the ribozymes A and B;

94 nucleotides between the ribozymes B and C*;

and 803 nucleotides from the ribozyme C* to the 3′ end.

The polyribozyme 161, 1076 bases long, is identical with thepolyribozyme 136, the only difference being that the ribozyme C atposition 204 possesses the G and the A deleted in the ribozyme C*.

The polyribozyme 163, 426 nucleotides long, contains the 3 ribozymes A(position 84), B (position 108) and C (position 204) surrounded bycomplementary arms of the following lengths:

82 nucleotides from the 5′ end to the ribozyme A;

22 nucleotides between the ribozymes A and B;

94 nucleotides between the ribozymes B and C;

and 153 nucleotides from the ribozyme C to the 3′ end.

The polykibozyme 165, 1099 nucleotides long, contains the 4 ribozymes A(position 84), B (position 108), C (position 204) and E (position 608)which are surrounded by complementary arms of the following lengths:

82 nucleotides from the 5′ end to the ribozyme A;

22 nucleotides between the ribozymes A and B;

94 nucleotides between the ribozymes C and E;

399 nucleotides from the ribozyme E to the 3′ end.

The polyribozymes do not contain the signals necessary for theirexpression and integration into the genome of the plants. A constitutiveor non-constitutive promoter (for example a viral or bacterial promotersuch as NOS, or a plant promoter such as that for Rubisco or that forubiquitin) must be placed at the 5′ end of the polyribozyme and apoly(A) sequence must be placed at the 3′ end. Several promoters whichfunction in plants can be used; the inventors selected the 35S promoterderived from the Cauliflower Mosaic Virus (CaMV), reputed to be thestrongest constitutive promoter. The polyadenylation signal poly (A) maybe that of the 35S gene of the CaMV, that of genes isolated from plantsor of octopine synthase; the inventors selected the poly (A) signalderived from the gene for nopaline synthase (tnos). The expressioncassettes constructed were introduced in the pBIOS 4 transformationvector derived from the pBI 121 vector containing the gene forresistance to kanamycin (gene neo which codes for neomycinphospho-transferase) and the iud A gene which codes for glucuronidase.The protocol for the genetic transformation of melon cotyledons and theregeneration of transgenic melon plants is identical with that describedin the patent “transgenic melon”, No. EP-A-0412912 in the name ofBIOSEM.

Example 1 Construction of a Ribosome Directed Against the Gene for theCapsid Protein of the Cucumber Mosaic Virus, Strain I17F

The phagemid bluescribe pBSIISK (STRATAGENE) containing the DNAcomplementary to the RNA4 of the CMVstrain I17F called pBIOS 113, theprocedure for the production of which is described in the Europeanpatent application EP-A-0412912, served as starting point for theconstruction of the different ribozymes used to obtain transgenic melonsresistant to different strains of CMV.

The catalytic site of the satellite TobRV (Tobacco Ringspot Virus) wasintroduced at different positions in the sequence of the gene for thecapsid protein. The sequence of the DNA complementary to the RNA4 of theCMV strain I17F, 1007 base pairs long, is shown in FIG. 3 ofEP-A-0412912; the part coding for the capsid protein is shown with theamino acid sequence.

As the ribozyme of the “Hammerhead” type described by Haselhoff andGerlach cleaves preferentially after the C of the GUC motifs, 4positions on the sequence coding for the capsid protein were selected(FIG. 2):

position A, nucleotide 84,

position B, nucleotide 108,

position C, nucleotide 204,

position E, nucleotide 608.

In order to introduce the 24 bases of the ribozyme commonly called“hammerhead” at these positions, it was decided to use the method ofmutagenesis on single-stranded DNA developed by KUNKEL (Proc. Nat. Acad.Sci. 82:488-492) which requires synthetic oligodeoxyribo-nucleotideswhich hybridize partially with the DNA that is to be mutagenised andwhich serve as primer at the 3′ end for the synthesis of a complementarystrand using a DNA polymerase. The following four oligonucleotides wereordered from the EUROGENTEC company:

Oligo A: 5′ TCGACGGTTACCTGATGAGTCCGTGAGGACGAAACCAGCACTGGTTG 3′ (SEQ. ID.NO:1) Oligo B: 5′ CGGGAACCACCTGATGAGTCCGTGAGGACGAAACGCGGACGACG 3′ (SEQ.ID. NO:2) Oligo C: 5′ GTTAATAGTTGCTGATGAGTCCGTGAGGACGAAACGACCAGCTGC 3′(SEQ. ID. NO:3) Oligo E: 5′GAATACACGAGCTGATGAGTCCGTGAGGACGAAACGGCGTACTTTC 3′ (SEQ. ID. NO:4)

With the aid of these 4 oligonucleotides and the directed mutagenesiskit purchased from the BIORAD company (catalogue number 170-3576) singlestranded DNA produced from the phagemid pBIOS 113 was mutagenised. FIG.3 shows the hybridization of the 4 oligonucleotides to the target singlestranded DNA and the arrows indicate the action of the DNA polymerasewhich synthesizes the complementary strand. The analysis of the fewrecombinant clones obtained after transformation of E. coli was firstmade by digestion with restriction enzymes in order to detect a sizeincrease of some of the fragments. One of the clones, pBIOS 116, whichapparently contained 3 catalytic sites was sequenced with the aid of the“sequenase R” kit version II obtained from the United StatesBiochemicals company by using an oligonucleotide of 22 bases, oligoNo.13 (position 53 to 74). The results showed the perfect insertion ofthe catalytic sites A and B and the imperfect insertion of the catalyticsite C. A deletion of the two bases at positions 20 and 21 (G and A) ofthe catalytic site had taken place. Although this site cannot befunctional for cleavage, as demonstrated by Lamb and Hay (Journal ofGeneral Virology, 1990, 71:2257-2264), it was decided to clone the DNAfragment of about 1100 bp (1007 bp+21 bp×2+19 bp+adjacent sequences ofthe polylinkers at 5′ and 3′) in opposite orientations in the expressionvector pBIOS 3 (Perez et al., Plant Mol. Biology 1989, 13:365-373), inorder to study the effect of the presence of non-complementary sequenceslacking ribozymic activity in the polyribozyme. For that purpose theKpnI-XbaI fragment of pBIOS 113 was cloned at the KpnI-XbaI sites of theplasmid pGEH7 2f (+) obtained from the PROMEGA BIOTECH company; this wasdone for the purpose of having BamHI sites on either side of thesequence for the capsid protein containing the catalytic sites. Theresulting plasmid pBIOS 151 was then digested by BamHI and the fragmentunder consideration (polyribozyme 136, FIG. 4) was introduced into theBamHI site of PBIOS 3. A recombinant clone containing the fragment inthe anti-sense orientation, under the control of the strong constitutivepromoter of the Cauliflower Mosaic Virus and the terminator of the genefor nopaline synthase, was selected and called pBIOS 125.

The EcoRI fragment of this plasmid containing the ribozyme(complementary fragment with two functional catalytic sites and onedeleted), under the control of the sequences for transcriptionalregulation mentioned above, was cloned at the EcoRI site of the binaryvector pBIOS 4. The latter is a derivative of the vector pBI 121(Jefferson et al., 1987: EMBO Journal 6:3901-3907) which was modified bythe suppression of the EcoRI site situated at the 3′ end of the genecoding for beta-glucuronidase and the creation of a EcoRI site situatedat the 5′ end of the same gene. The resulting binary vector, pBIOS 136,was used in different transformation experiments after triparentalconjugation in the disarmed strain of Agrobacterium tumefaciens RC58′3,which is derived from the strain C58′3 (Mullineaux et al., Plant Science63:237-245, 1989) and is in fact a spontaneous mutant resistant torifampicin.

Example 2 Construction of Differents Ribozymes Directed Against the Genefor the Capsid Protein of the Cucumber Mosaic Virus, Strain I17F

Given that the polyribozyme 136 (FIG. 4) described in Example 1 did notcontain the catalytic site directed against position 608 and that theone directed against position 204 was incomplete, further directedmutagenesis experiments were initiated. To this end, the EcoRI fragmentof the plasmid was cloned in the phage M13 mp18 digested by EcoRI,obtained from the PHARMACIA company. A recombinant phage allowing theencapsidation of the coding strand of the gene for the capsid proteincontaining the two correct catalytic sites was characterised and it wasused as matrix for the new mutagenesis experiments utilizing theoligodeoxyribonucleotides C and E (together or separately). These latterwere conducted like those presented in the previous example, except thatthe yield of single stranded matrix is much more favourable since thestarting material was a phage. Different recombinant clones weresequenced by using the oligonucleotide No.13 and a 19-meroligonucleotide (position 694 to position 676), this latter making itpossible to sequence the catalytic site introduced at position 608. Theclones containing catalytic sites in conformity with the invention wereused for the construction of the binary vectors pBIOS 161, pBIOS 163 andpBIOS 165 (see FIG. 4). The binary vector pBIOS 161 is identical withpBIOS 136 except that pBIOS 161 contains the catalytic site C undeleted,

In the case of genes coding for the ribozymes which hybridize with theentire sequence of the gene for the capsid protein and containing threefunctional catalytic sites A, B, C (the case for pBIOS 161) or fourfunctional catalytic sites A, B, C, E (the case for pBIOS 165), cloningat the EcoRI site of the binary vector pBIOS 4 is carried out directlyafter purification of the EcoRI fragment comprising the 35S promoter,the ribozyme, the terminator NOS. In the case of the ribozyme whichcontains 3 catalytic sites A, B, C and hybridizes with only the first360 bases at the 5′ end of the RNA4 of the C4V (the case for pBIOS 163),a deletion of the remaining 3′ part was made after digestion with therestriction enzyme HindIII (positions 361 in the sequence of the genefor the capsid protein and of the HindIII site situated at the 3′ borderof this sequence and resulting from a polylinker). The thus deletedEcoRI fragment of the plasmid was then cloned at the EcoRI site of thebinary vector pBIOS 4.

Transformation and Regeneration of Melons Expressing the Polyribozymes

The last 3 binary vectors were introduced into Agrobacterium and usedfor transformation as described in the patent application EP-A-0412912.

Example 3 Molecular Analysis of the Transgenic Plants

Northern Blot Analyses:

The melon plants obtained after transformation with pBIOS 136 wereanalysed by means of Northern blot (FIG. 5). The total RNAs wereextracted from young leaves of transgenic and non-transgenic plantscultivated in a greenhouse, according to the protocol of Chandler et al.(Plant Physiology (1983) 74:47-54).

They were subjected to electrophoresis in a 1% denaturing agarose gel,transferred to Hybond C and hybridized with the probe constituted by thefragments resulting from a triple digestion of the BamHI fragment, whichcorresponds to the complete complementary fragment of the gene for thecapsid protein, bearing the 3 ribozymes, two of which are functional(polyribozyme 136). The triple digestion favours hybridization betweenthe homologous sequences and makes it possible to obtain a more intensesignal.

The homogeneity of the quantities of total RNAs loaded on to the gel andthe quality of the RNAs were verified by staining of the membrane withmethylene blue. The results obtained at the transcriptional level forthe primary transformants and the corresponding T1 and T2 descendantsare illustrated in FIG. 2 and show that:

a major transcript of 1.45 kb is observed in all of the samples derivedfrom transformed plants but not in the negative control;

the number of transcripts varies considerably according to the primarytransformants. This may be explained by integrations of the T-DNA orfragments of T-DNA at different loci of the plant genome and thus byenvironmental effects:

no correlation exists between the transcriptional levels of the primarytransformants and those of their T1 and T2 descendants.

Southern Blot Analyses

The melon plants obtained after transformation with pBIOS 136 or pBIOS135 were analysed by means of Southern blot (FIGS. 6 and 7). The totalDNAs were extracted from young leaves of transgenic (primarytransformants, T1 and T2 descendants in some cases) and non-transgenicplants cultivated in a greenhouse, according to the protocol ofDellaporta et al. (Plant Molecular Biology Reporter (1983) 1:19-21).They were hydrolysed by EcoRI (cloning site of the expression cassetteof the gene of interest in pBIOS4), subjected to electrophoresis in 0.8%agarose gel, transferred to Hybond N+ and hybridized with the threeprobes, gene npt II, polyribozyme 136 triply digested (cf. Northern blotanalysis) and gene gus.

The results obtained for 5 transformation events with PBIOS 136 (FIG. 6)show that:

Lines 146.42:

In the case of the primary transformant 146.42, its T1 descendant andtwo T2 descendants, the hybridization profiles with the 3 probes areidentical, which indicates that there has been no segregation of thefragments of the T-DNA and that there is probably a single locus Thepolyribozyme 136 is present in the plant genome in several copies. Infact, 4 hybridization bands with sizes of 1.75 kb; 4.4 kb; 8.3 kb and8.8 kb are visible. The 1.75 kb band corresponds to the theoretical sizeof the band expected with the couple (EcoRI, polyribozyme). The 4.4 kbband hybridizes with both the polyribozyme 136 and the gene npt II.Furthermore, the analysis with the EcoRI/npt II couple does not lead tothe detection of this band, which indicates the presence of a singlecopy of the npt II gene in the plant genome. The bands of 8.3 kb and 8.8kb hybridize with the polyribozyme 136 and the gus gene. Only these twobands are detected by the EcoRI/gus couple, which reveals the presenceof two copies of all or part of the gus gene. The hybridization of the 3probes with DNA of the T0, T1 and T2 plants digested with XbaI alsosuggests the presence of a single locus.

Lines 146.34:

In the case of the primary transformant 146.34 and its T1 descendant,only some of the hybridization bands of the primary transformant subsistin the individual T1, which emphasizes the fact that certain fragmentsof the T-DNA have been eliminated. For the analysis of theEcoRI/polyribozyme 136, 3 bands with sizes of 1.75 kb, 3 kb and 5.3 kbare common to the T0 and T1 plants and 3 additional bands with sizes of7.3 kb, 6.8 kb and 4.25 kb characterize the plant T0. This indicates thepresence of at least 3 and 6 copies of the polyribozyme 136 in the T1and T0 plants, respectively. In npt II hybridization, the two EcoRIbands of 3 kb and 5.3 kb are detected in the T0 plants, which shows thepresence of two copies of all or part of the npt II gene. Only the EcoRIband of 3 kb is visible in the T1 plant, which indicates the presence ofa single copy of the npt II gene. In gus hybridization, the 3 EcoRIbands with sizes of 4.25 kb, 5.3 kb and 6.8 kb are found in the T0 plantwhereas only the band of 5.3 kb is present in the T1 descendant. Thisreveals the presence of 3 copies and one copy of all or part of the gusgene in the T0 and T1 plants, respectively. Moreover, the hybridizationof the 3 probes with the DNAs of the T0 and T1 plants digested by XbaIsuggests the existence of 2 loci.

Lines 146.30 and 146.28:

In the case of the primary transformants 146.30 and 146.28 as well astheir respective T1 descendants similar observations are made.

Line 141.1:

In the case of the primary transformant 141.1 and its descendant, thehybridization profiles are similar. EcoRI bands of 1.75 kb, 10 kb and 10kb are revealed with the probes polyribozyme 136, npt II and gus,respectively. This shows the presence of a single copy of the T-DNAintegrated into the plant genome.

In conclusion, genetic stability is observed in the case of thetransformant 146.42 and its descendants (T1 and T2) and in that of thetransformant 141.1 and its descendant T1. A high number of copies of theT-DNA or fragments of the T-DNA characterise 4 of the lines studiedwhereas the line 141.1 possesses only one integrated copy of the T-DNA.

Moreover, the presence of a copy of the T-DNA and/or part of the T-DNAis also to be noted in the lines transformed with pBIOS 135 (FIG. 7).

Western Blot Analysis (Comparative Analysis)

The transgenic melon plants transformed with pBIOS 4 containing theexpression cassette of the gene for the capsid protein were analysed bymeans of Western blot. The patent application EP-A-0412912 of the11.08.1989 in the name of BIOSEM recounts the methodology employed andthe results obtained. However, it should be emphasized that the level ofexpression of the gene for the capsid protein varies between 0.001% and0.4% of the total proteins. By means of a few examples, FIG. 8illustrates this variation in the level of expression, which depends onthe age of the leaf and probably on environmental effects on the plantgenome.

Example 4 Tests of Resistance of the Melons Transformed by thePolyribozyme 136 and the Capsid Protein to the CMV (COMPARATIVE TEST)

The transgenic melon plants (genotype TEZIER 10) which express the genefor the capsid protein or the polyribozyme 136 were self-fertilized orcrossed with untransformed plants of the genotype TEZIER 10. The seedsderived from these self-fertilizations (T1 generation) and subsequentself-fertilizations (generation T2, . . . ) were sown in a phytotron(climatised chamber with a 16 hour light period).

At the 2 to 4 leaves stage, 23 plants originating from the descendantswere mechanically inoculated with a powdered preparation of fresh leavesinfected with the CMV strain TL28.

After 16 days of infection (optimal period), the symptoms (mosaic,pleating of the leaf blade, yellowing) were evaluated. The infectedplants are classified in three category;

resistant plants (R) which have no symptoms;

sensitive plants (S) which have symptoms;

tolerant plants (T) which are the plants which “recover”, i.e. thenascent leaves develop without symptoms whereas the old leaves exhibitsymptoms.

Several cycles of “recovery” may occur. Whether or not nascent healthyleaves change into infected leaves depends on the climatic conditions.

The “resistant-type” plants include both the plants without symptoms andtolerant plants.

The controls used in the test of resistance to CMV are:

sensitive controls: TEZIER 10 and Vedrantais;

resistant controls which possess at least three recessive genes of theCMV, Virgos and Free Cucumber.

Resistance of Melon Lines in Generation T1 Which Express the Gene forthe Capsid Protein to the CMV Strain TL 28:

The results obtained for 20 lines are given in Table 1 and show that:

The infection with TL 28 leads to the production of 3 to 30% of TIplants without symptoms. The untransformed control TEZIER 10, inoculatedwith TL 28 yields 0% of plants without symptoms. The resistant controlsVirgos and Free Cucumber give rise to 92 and 100% of plants withoutsymptoms, respectively, and do not show any tolerance phenomenon.

The phenomenon of recovery exists in a high proportion of cases.Fourteen lines studied show a not insignificant percentage (22 to 59%)of tolerant T1 plants.

There is no correlation between the percentage of resistant and tolerantT1 plants and the level of expression of the capsid protein. In fact,the lines 159.8 and 164.2, for example, which have an identical level ofexpression of the capsid protein (0.01%) exhibit different levels ofresistance. The T1 individuals of the 159.8 line are all sensitivewhereas in the 164.2 line 26% are without symptoms and 48% are tolerant.

Most of the lines expressing the gene for the capsid protein wereobtained by self-fertilization. The expected theoretical frequency ofthe gene for the capsid protein is 75% in the T1 plants inoculated withthe CMV. The percentage of resistant-type plants (without symptoms andtolerant) vary between 25 and 82% depending on the transgenic lines.

TABLE 1 RESISTANCE OF T1 GENERATION MELON PLANTS WHICH EXPRESS THE GENEFOR THE CAPSID PROTEIN TO THE CMV STRAIN TL28 LINES CROSS % CP % R % T %S 88.105 I 0.01 3 22 75 131.6 BC 0.06 9 0 81 145.1 I 4 0 96 145.2 BC0.06 22 35 43 153.4 I 0.1 0 30 70 153.5 I 0.06 17 57 26 153.6 I 0.13 1832 50 153.8 I 0.01 17 43 40 153.9 I 0.06 30 39 31 153.19 I 0.05 29 52 19153.20 I 0.01 30 39 31 153.22 I 0.4 18 27 55 159.4 BC 0.002 0 0 100159.8 I 0.01 0 0 100 164.2 I 0.01 26 48 26 164.23 BC 0.01 30 35 35 166.5I 23 55 22 166.10 I 23 59 18 170.5 BC 0.3 9 0 91 171.7 I 9 0 91 TEZIER10 0 0 100 VEDRANTAIS 0 0 100 VIRGOS 92 0 8 FREE CUCUMBER 100 0 0

Legend:

I: self-fertilization

BC: cross with the untransformed genotype TEZIER 10

% CP: level of expression of the capsid protein as percentage of thesoluble total proteins

% R: percentage of resistant plants or plants without symptoms

% T: percentage of tolerant plants

% S: percentage of sensitive plants

Resistance of Melon Lines in Generation T1 Which Express thePolyribozyme 136 to the CMV Strain TL 28:

The results obtained for 13 lines are given in Table 2 and show that:

in the case of 10 lines, 30 to 87% of the T1 individuals are withoutsymptoms after infection with TL 28;

the “recovery” phenomenon is scarcely present. Only 4 lines showtolerant T1 plants, to the extent of 9 to 26%.

Most of the lines expressing the polyribozyme 136 were obtained bycrosses with the untransformed line TZ 10. The expected theoreticalfrequency of the polyribozyme is 50% in the T1 plants inoculated withthe CMV. The percentage of “resistant-type” plants varies between 30 and65% depending on the transgenic lines.

In conclusion, in the case of the “ribozyme” strategy, a larger numberof lines of the T1 generation are without symptoms and possess very fewtolerant plants.

TABLE 2 RESISTANCE OF T1 GENERATION MELON PLANTS WHICH EXPRESS THEPOLYRIBOZYME 136 TO THE CMV STRAIN TL28 LINES CROSS % R % T % S 141.1 BC48 9 43 141.2 BC 39 26 35 141.3 BC 43 17 40 141.4 BC 26 26 48 141.5 BC52 0 48 141.6 BC 39 0 61 146.19 BC 30 0 70 146.28 BC 47 0 53 146.42 I 870 13 146.47 BC 43 0 57 TEZIER 10 0 0 100 VEDRANTAIS 0 0 100 VIRGOS 92 08 FREE CUCUMBER 100 0 0

Legend:

I: self-fertilization

BC: cross with the untransformed genotype TEZIER 10

% R: percentage of resistant plants or plants without symptoms

% T: percentage of tolerant plants

% S: percentage of sensitive plants

Summary of the Tests of Resistance to the CMV Strain TL 28 and of theEvaluation of Segregation by Means of the GUS Test for Certain Lines:

The results presented in Table 3 show:

the production of two lines of the resistant type (plants withoutsymptoms and tolerant plants) which express the gene for the capsidprotein (lines 88.105 and 153.8) and one completely resistant line(plants without symptoms) which expresses the polyribozyme 136 (line146.42).

the production of two tolerant lines (tolerant plants) which express thepolyribozyme 136 (lines 141.1 and 146.28).

TABLE 3 TEST OF RESISTANCE TO THE CMV STRAIN TL28 AND BEHAVIOURS OF THEGUS GENE AND THE GENE FOR RESISTANCE IN THE DESCENDANTS MOLEC. R T SLINES X GEN CHARACT. % G % R % G QT. VIRUS % R % G QT. VIRUS % R % G QT.VIRUS 88.105 T0 K.CP.G. 702 I T1 K.CP.G. 80 3 100 0.78 +/− 22 100 1.46+/− 75 53 1.42 +/− 0.02 0.39 0.25 710 I T2 K.CP.G. 100 100 100 0.71 +/−0.11 153.8 T0 K.CP.G. 740 I T1 K.CP.G. 73 12 100 0.56 +/− 43 100 0.85+/− 45 55 0.94 +/− 0.10 0.10 0.17 545 I T2 K.CP.G. 100 50 100 0.69 +/−50 100 0.92 +/− 0.56 0.57 159.8 T0 K.CP.G. 739.1 I T1 K.CP.G. 0 100 01.42 +/− 0.27 141.1 T0 K.RZ.G. 730.1 BC T1 K.RZ.G. 57 40 100 0.88 +/− 6019 1.05 +/− 0.21 0.17 146.28 T0 K.RZ.G. 734.1 BC T1 K.RZ.G. 73 100 731.96 +/− 0.46 539.2 I T2 K.RZ.G. 68 37 100 1.00 +/− 63 53 1.64 +/− 0.250.42 146.31 T0 K.RZ.G 735.1 BC T1 K.RZ.G 0 100 0 1.64 +/− 0.46 146.42 T0K.RZ.G. 755.1 I T1 K.RZ.G. 77 79 100 0.42 +/− 21 0 1.75 +/− 0.22 0.37540 I T2 K.RZ.G. 82 90 100 0.45 +/− 10 0 1.52 +/− 0.30 0.65 TEZIER 10 00 100 1.70 +/− 0.77 VEDRANTAIS 0 0 100 1.70 +/− 0.72 VIRGOS 96 0.05 +/−4 0 0.02 FREE CUCUMBER 100 0.21 +/− 0 0 0.10

Legend:

X: type of cross; I: self-fertilization; BC: cross with theuntransformed genotype TEZIER 10

GEN: generation; T0: primary transformant; T1: No.1 descendant; T2: No.2descendant.

K: nptII gene; CP: capsid protein gene; RZ: polyribozyme 136; G: gusgene

+: presence; −: absence; QT: quantity

% G: percentage of plants expressing the gus gene

% R: percentage of resistant plants or plants without symptoms

% T: percentage of tolerant plants

% S: percentage of sensitive plants

1) Genetic Study of the Lines:

The level of expression of the gus gene enables the type of segregationand the state of homozygosity to be determined.

In the case of the two lines 88.105 and 153.8, the level of expressionof the gus gene is 80% and 73%, respectively, in the T1 generationobtained by means of self-fertilization. This indicates a Mendelian typeof segregation of a dominant gene (3:1) with the integration of theT-DNA at a single locus. Molecular analyses have shown the presence of asingle copy of the T-DNA integrated into the plant genome.

Furthermore, the level of expression of the gus gene is 100% in the T2generation for both lines, which confirms the state of homozygosity ofthe integrated gene.

In the case of line 146.42, the level of expression of the gus gene is77% in the T1 generation obtained by self-fertilization, which shows aMendelian type of segregation of a dominant gene with integration of theT-DNA at a single locus. Molecular analyses have shown the presence ofseveral T-DNA and/or T-DNA fragments at a single locus of the plantgenome.

Furthermore, the fact that the level of expression of the gus gene is90% in the T2 generation emphasizes that the line tested is nothomo-zygous for this integrated gene (Table 3). Homozygosity wasobtained in T3 in the case of line 146.42. The level of expression ofthe gus gene for line 141.1 is 57% in the T1 generation obtained aftercrossing with the untransformed TEZIER 10.genotype. This alsocorresponds to a Mendelian type of segregation of a dominant gene (1:1)with integration of the T-DNA at a single locus. Molecular analyses haveshown the presence of a single T-DNA integrated into the plant genome.

In the case of line 146.28, the level of expression of the gus gene is73% in the T1 generation obtained after crossing with the untransformedTEZIER 10 genotype. This indicates a segregation of the dominant geneswith integration of the T-DNA at several loci. Molecular analyses haveshown the presence of several T-DNA or T-DNA fragments at two loci ofthe plant genome.

2) Behaviour vis-a-vis the Virus:

All of the resistant-type plants express the gus gene. Some of thesensitive plants express the gus gene (Table 3).

In the case of line 88.105, 25% of plants of the resistant type and 75%of sensitive plants were obtained in the T1 generation as a result ofself-fertilization. This shows a Mendelian type of segregation of arecessive gene (1:3) for the resistance gene.

In the case of line 153.8, 55% of plants of the resistant type and 45%of sensitive plants were obtained in the T1 generation as a result ofself-fertilization. It is difficult to come to a conclusion with regardto the resistance gene.

In the case of line 141.1, 40% of tolerant plants and 60% of sensitiveplants were obtained in the T1 generation by crossing with theuntransformed TEZIER 10 genotype. This indicates a Mendelian type ofsegregation of a dominant gene (1:1) for the resistance gene. Thebehaviour of the polyribozyme 136 is thus similar to that of the gusgene.

In the case of line 146.42, 79% of plants without symptoms and 21% ofsensitive plants were obtained in the T1 generation as a result ofself-fertilization. This implies a Mendelian type of segregation of adominant gene (3:1) for the resistance gene. The behaviour of theribozyme 136 is thus similar to that of the gus gene.

3) Estimation of the Quantities of Virus:

The quantities of virus detected by the ELISA assay are quite low in theplants without symptoms but nonetheless higher than those in the FreeCucumber resistant control (Table 10).

The tolerant plants contain appreciable quantities of virus.

The sensitive plants possess very high quantities of virus.

The plants expressing the polyribozymes (lines 146.42, T2 and T3)contain a smaller amount of virus than the plants of the resistant typeexpressing the capsid protein (line 153.8, T2).

It appears that there is a quite good correlation between the quantitiesof virus detected and the severity of the symptoms.

4) Development of the Symptoms of the CKV With Time:

T1 Plants of the lines mentioned in FIGS. 1 and 2 were infected with theCMV strain TL 28. The symptoms were recorded 6, 8, 12, 14, 16 and 19days after inoculation. The results are presented in the histograms(FIGS. 9 and 10).

The values obtained after D16 show no further variation. In the case ofthe “capsid protein” lines, a more or less rapid diminution of thenumber of plants without symptoms is to be noted in favour of theappearance of plants with symptoms. In the case of the lines 88-105 and153-8 in the T1 generation, on D16 and D14 respectively, tolerant plantsdevelop characterized by the phenomenon of “recovery”. Furthermore, inline 88-105 in the T2 generation, 100% of the plants without symptoms onD8 are converted into 100% of tolerant plants which subsist until D19(FIG. 9).

This sudden change of the plants without symptoms into tolerant plantsmay be explained by a pronounced effect of the climatic conditions.

In line 153.8 in the T2 generation, a progressive appearance of tolerantplants is to be noted. From D16 onwards, 50% of the T2 plants arewithout symptoms and 50% are tolerant.

In the case of the “polyribozyme” lines, a diminution of the number ofT1 plants without symptoms in favour of plants with symptoms is to beemphasized in lines 141.1 and 146.28 (FIG. 10).

Furthermore, in the case of line 141.1, 40% of tolerant T1 plants is tobe noted from D16 onwards. In line 146.28, tolerant T2 plants developfrom D14 to reach 26% on D16.

Moreover, in line 146.42, the T1 plants without symptoms represent 80%on D6 and remain almost stable with time (78% on D8, D12 and D14, 79% asof D16).

The T2 plants of line 146.42 exhibit a similar behaviour. This resultshows the very high level of resistance of this line and stability ofthe resistance gene in the descendants.

In conclusion, two lines of the “resistant” type which express the genefor the capsid protein and one completely resistant line which expressesthe polyribozyme 136 were obtained. In the case of the two linesexpressing the gene for the capsid protein the resistance observed ismore similar to tolerance than to complete resistance. In fact, someplants infected by the virus which exhibit severe symptoms are able todevelop new healthy leaves under certain conditions.

No tolerance phenomenom is observed in the case of the completelyresistant line which expresses the polyribozyme 136. In the case of the2 lines which express the capsid protein, relatively high quantities ofvirus were observed in the resistant plants, whereas in the resistantplants which express the polyribozyme 136 the quantity of virus isalmost the same as in the Free Cucumber resistant control.

Example 5 Resistance of Melon Lines in Generation T1, Which Express thePolyribozyme 165, to Infection by CMV Strain TL28:

The results obtained for 13 T1 lines expressing the polyribozyme 165,are presented in table 4 and show that:

after infection with TL28, 15 to 100% of the T1 individuals are withoutsymptoms for 12 lines;

the phenomenom of “recovery” is only slightly present. Only 5 lines havebetween 6 and 60% T1 tolerants plants.

The majority of the lines expressing the polyribozyme 165 have beenobtained by self-fertilisation. The expected theoretical frequency ofpolyribozyme 165 is 75% in T1 plants inoculated with CMV. The percentageof “resistant-type” plants varies between 15 and 100% according to thetransgenic line.

In conclusion, as with the lines expressing the polyribozyme 136, alarge number of lines expressing the polyribozyme 165 are withoutsymptoms and have few tolerant plants. The polyribozyme 165 differs fromthe polyribozyme 136 by two additional functional ribozymes. The twoconstructions give similar results for resistance to infection by CMV.

TABLE 4 RESISTANCE OF T1 GENERATION MELON PLANTS EXPRESSING THEPOLYRIBOZYME 165 TO INFECTION BY CMV STRAIN TL28 LINES CROSS % R % T % S10.2 I 100 0 0 203.6 BC 0 0 100 205.1 I 50 45 5 205.3 I 66 0 34 206.1 I94 6 0 207.3 I 61 0 39 207.5 I 80 0 20 207.7 I 64 0 36 207.8 I 69 9 22207.9 I 100 0 0 211.1 I 15 0 85 212.1 I 87 13 0 215.1 I 20 60 20VEDRANTAIS 0 0 100 VIRGOS 100 0 0 FREE 100 0 0 CUCUMBER

Legend:

I: self-fertilization

BC: cross with the untransformed genotype TELIZIER 10

% R: percentage of resistant plants or plants without symptoms

% T: percentage of tolerant plants

% S: percentage of sensitive plants

14 1 47 DNA Artificial Sequence Description of ArtificialSequenceSynthetic ribozymes and portions thereof 1 tcgacggtta cctgatgagtccgtgaggac gaaaccagca ctggttg 47 2 44 DNA Artificial SequenceDescription of Artificial SequenceSynthetic ribozymes and portionsthereof 2 cgggaaccac ctgatgagtc cgtgaggacg aaacgcggac gacg 44 3 45 DNAArtificial Sequence Description of Artificial SequenceSyntheticribozymes and portions thereof 3 gttaatagtt gctgatgagt ccgtgaggacgaaacgacca gctgc 45 4 46 DNA Artificial Sequence Description ofArtificial SequenceSynthetic ribozymes and portions thereof 4 gaatacacgagctgatgagt ccgtgaggac gaaacggcgt actttc 46 5 1007 DNA ArtificialSequence Description of Artificial SequenceSynthetic ribozymes andportions thereof 5 agagagtgtg tgtgctgtgt tttctctttt gtgtcgtagaattgagtcga gtcatggaca 60 aatctgaatc aaccagtgct ggtcgtaacc gtcgacgtcgtccgcgtcgt ggttcccgct 120 ccgccccctc ctccgcggat gctaacttta gagtcttgtcgcagcatctt tcgcgactta 180 ataagacgtt agcagctggt cgtccaacta ttaaccacccaacctttgta gggagtgaac 240 gctgtagacc tgggtacacg ttcacatcta ttaccctaaagccaccaaaa atagaccgtg 300 ggtcttatta cggtaaaagg ttgttactac ctgattcagtcacggaatat gataagaagc 360 ttgtttcgcg cattcaaatt cgagttaatc ctttgccgaaatttgattct accgtgtggg 420 tgacagtccg taaagttcct gcctcctcgg acttatccgttgccgccatc tctgctatgt 480 tcgcggacgg agcctcaccg gtactggttt atcagtatgccgcatctgga gtccaagcca 540 acaacaaact gttgtatgat ctttcggcga tgcgcgctgatataggtgac atgagaaagt 600 acgccgtcct cgtgtattca aaagacgatg cgctagagacggacgagcta gtacttcatg 660 ttgacatcga gcaccaacgc attcccacgt ctggagtgctcccagtctga ttcgtgttcc 720 cagaatcctc cctccgatct ctgtggcggg agctgagttggcagttctgc tataaactgt 780 ctgaagtcac taaacgtttt tacggtgaac gggttgtccatccagcttac ggctaaaatg 840 gtcagtcgtg gagaaatcca cgccagtaga tttacaaatctctgaggcgc ctttgaaacc 900 atctcctagg tttcttcgga aggacttcgg tccgtgtacctctagcacaa cgtgctagtt 960 tcagggtacg ggtgcccccc cactttcgtg ggggcctccaaaaggag 1007 6 33 DNA Artificial Sequence Description of ArtificialSequenceSynthetic ribozymes and portions thereof 6 tctgaatcaa ccagtgctggtcgtaaccgt cga 33 7 47 DNA Artificial Sequence Description of ArtificialSequenceSynthetic ribozymes and portions thereof 7 gttggtcacg accaaagcaggagtgcctga gtagtccatt ggcagct 47 8 27 DNA Artificial SequenceDescription of Artificial SequenceSynthetic ribozymes and portionsthereof 8 cgtcgtccgc gtcgtggttc ccgctcc 27 9 44 DNA Artificial SequenceDescription of Artificial SequenceSynthetic ribozymes and portionsthereof 9 gcagcaggcg caaagcagga gtgcctgagt cgtccaccaa gggc 44 10 33 DNAArtificial Sequence Description of Artificial SequenceSyntheticribozymes and portions thereof 10 gttagcagct ggtcgtcaac tattaaccac cca33 11 45 DNA Artificial Sequence Description of ArtificialSequenceSynthetic ribozymes and portions thereof 11 cgtcgaccagcaaagcagga gtgcctgagt agtcgttgat aattg 45 12 35 DNA Artificial SequenceDescription of Artificial SequenceSynthetic ribozymes and portionsthereof 12 acatgagaaa gtacgccgtc ctcgtgtatt caaaa 35 13 46 DNAArtificial Sequence Description of Artificial SequenceSyntheticribozymes and portions thereof 13 ctttcatgcg gcaaagcagg agtgcctgagtagtcgagca cataag 46 14 36 RNA Artificial Sequence n=g, a, c or t(u) 14nnnnnncuga ugaguccgug aggacgaaac nnnnnn 36

What is claimed is:
 1. A polyribozyme having endoribonuclease activityand which inactivates the capsid protein gene of a plant virus, whereinthe polyribozyme comprises a plurality of ribozymes, each of whichcleaves the capsid protein gene or its corresponding transcript, or itscorresponding replication intermediate, wherein each ribozyme comprisesa catalytic portion and two hybridizing arms that are complementary to aportion of the capsid protein gene of the plant virus, wherein the plantvirus is selected from the group consisting of: the Caulimoviruses, theGeminiviruses, the Reoviridae, the Rhabdoviridae, the Tobamoviruses, thePotexviruses, the Potyviruses, the Carlaviruses, the Closteroviruses,the Tobraviruses, the Hordeiviruses, the Tymoviruses, the Barley YellowDwarf Virus, the Tombusviruses, the Sobemoviruses, the Neopviruses, theComoviruses, the Cucumoviruses, the Bromoviruses, and the Ilarviruses.2. The polyribozyme according to claim 1 having the formula:[(Y₁—Q—Y₂)—(S)_(n)]_(p) wherein Y₁ and Y₂ are each a hybridizing armhaving at least 4 bases and being complementary to a part of the capsidprotein gene, a part of its corresponding transcript, or a part of itscorresponding replication intermediate; Q is the catalytic region of aribozyme; S is a nucleotide sequence which has between 2 and 500 basesand is not complementary to the capsid protein gene, its correspondingtranscript or its corresponding replication Intermediate; n=0 or 1; andp is an integer greater than
 1. 3. The polyribozyme according to claim 1wherein each ribozyme as selected from the group consisting of ahammerhead ribozyme and a hairpin ribozyme.
 4. The polyribozymeaccording to claim 1 wherein each ribozyme targets an XUX site.
 5. Thepolyribozyme according to claim 4 wherein the number of ribozymes isequal to or less than the total number of XUX sites present in thecapsid protein gene, or in its corresponding transcript, or itscorresponding replication intermediate.
 6. The polyribozyme according toclaim 5, wherein each ribozyme targets an XUX site which occurs in azone of homology conserved between different strains of the same virusor between different viruses.
 7. The polyribozyme according to claim 1,wherein the plant virus is selected from the group consisting of:Cauliflower Mosaic Virus (CaMV), Maize Streak Virus (MSV) Wound TumorVirus (WTV), Potato Yellow Dwarf Virus (PYDV), Tomato Spotted Wilt Virus(TSWV), Tobacco Mosaic Virus (TMV), Potato Virus X (PXV), Potato Virus Y(PYV), Carnation Latent Virus (CLV), Beet Yellow Virus (BYV), TobaccoRattle Virus (TRV), Barley Stripe Mosaic Virus (BSMV), Turnip YellowMosaic Virus (TYMV), Barley Yellow Dwarf Virus (BYDV), Tomato BushyStunt Virus (TBSV), Southern Bean Mosaic Virus (SBMV), Tobacco NecrosisVirus (TNV), Tobacco Ring Spot Virus (TRSV), Cow Pea Mosaic Virus(CPMV), Pea Enation Mosaic Virus (PEMV), Cucumber Mosaic Virus (CMV),Brome Mosaic Virus (BMV), and Tobacco Streak Virus (TSV).
 8. Thepolyribozyme according to claim 7, wherein each hybridizing arm iscomplementary to the transcript of the capsid protein gene of theCucumber Mosaic Virus.
 9. The polyribozyme of claim 1 consisting of RNA.10. A DNA molecule coding for a polyribozyme according to claim
 1. 11. Aprocess for rendering a slant resistant to a virus, which processcomprises the introduction into the plant of the DNA of claim
 10. 12.The process according to claim 11, wherein the introduction into theplant of he DNA is made by genetic transformation of a part of the plantby a DNA molecule coding for the polyribozyme, followed by regenerationof a transgenic plant.
 13. The process according to claim 12, whereinthe transformation is carried out by the intermediary of Agrobacteriumtumefaciens or Agrobacterium rhizogenes.
 14. A transgenic plantresistant to a virus, said plant containing in its genome a sequencewhich gives rise on transcription to the polyribozyme according toclaim
 1. 15. The transgenic plant according to claim 14, the plant beingresistant to Cucumber Mosaic Virus.
 16. The transgenic plant accordingto claim 15 which is a melon, a cucumber, a courgette, a tomato, a sweetpepper or a bean.
 17. Transgenic fruit of the plant according to claim14.
 18. Transgenic fruit of the plant according to claim
 15. 19.Transgenic seed of the plant according to claim
 14. 20. Transgenic seedof the plant according to claim
 15. 21. A plant cell transformed by theDNA according to claim 10.